The analysis of circulating cell-free DNA has been increasingly used for the detection and monitoring of cancers (1-3). Different cancer-associated molecular characteristics, including copy number aberrations (4-7), methylation changes (8-11), single nucleotide mutations (4, 12-15), cancer-derived viral sequences (16, 17) and chromosomal rearrangements (18, 19) can be detected in the plasma of patients with various types of cancers. Despite the rapid expansion of clinical applications, many fundamental molecular characteristics of circulating DNA in cancer patients remain unclear, thereby limiting the most effective clinical use of such analyses.
In particular, previous studies on the size of circulating DNA in cancer patients gave inconsistent results. Studies have demonstrated that the overall integrity (a measurement of size) of circulating DNA would increase in cancer patients when compared with subjects without a malignant condition (20-23). Using PCR with different amplicon sizes, it was shown that the proportion of longer DNA would be higher in cancer patients. This aberration in DNA integrity was shown to be reversible after treatment and the persistence of such changes was associated with poor prognosis (20, 24). On the other hand, there is also seemingly contradictory evidence that circulating DNA derived from tumor tissues might be shorter than those derived from non-malignant cells. For example, it has been shown that the proportion of DNA molecules carrying cancer-associated mutations would be higher when those mutations were detected using PCR with shorter amplicons (12, 25).
Further, studying the size profile of tumor-derived DNA in the plasma of the HCC patients is a challenging endeavor because tumor-derived plasma DNA cannot be readily distinguished from the non-tumor-derived background DNA in plasma. The detection of cancer-specific mutations offers a genotypic means to distinguish the tumoral from the non-tumoral plasma DNA. However, there are relatively few cancer-specific mutations across the genome (29-32). Accordingly, it can be difficult to accurately identify tumor-derived DNA in plasma, particularly for the purpose of generating a broad, detailed and yet cost-effective view of the size distribution of tumor-derived DNA.
Such difficulties provide obstacles in obtaining accurate measurements in samples possibly containing mixtures of tumoral and non-tumoral DNA.